Inhibition of cell proliferation by C/EBPalpha, a nuclear transcription factor expressed at high levels in hepatocytes, is well documented, however, the molecular mechanisms of C/EBPalpha- mediated growth arrest are unknown. We have shown that C/EBPalpha inhibits cell proliferation through p21/SDI-1 protein. This project is focused on the study of the C/EBPalpha dependent regulation of p21/SDI-1 in vivo and on the investigation of this growth inhibitiory, pathway in regenerating hepatocytes in young and old rats. Preliminary data show that is hepatocytes, C/EBPalpha controls the level of p21/SDI-1 protein and that this control is altered in the livers of old rats. In Specific Aim 1, we will investigate the regulation of P21/SDI-1 expression in regenerating rat liver. A correlative reduction of both C/EBPalpha and p21/SDI-1 would suggest that C/EBPalpha inhibits hepatocyte growth in vivo via the p21/SDI-1 protein. Expression of other cyclin dependent kinase (CDK) inhibitors during liver regeneration will be investigated to determine the specificity of C/EBPalpha regulation of p21/SDI-1. We will also study regeneration in p21 knock-out mice. If C/EBPalpha inhibits liver growth through p21/SDI-1, it is likely that changes in the pattern regeneration will be detected in p21 knock-out liver following partial hepatectomy. We will use another genetic knockout model (for the C/EBPa gene) to investigate the developmental expression of C/EBPalpha and p21 in both wild type and C/EBPalpha null mice. In Specific Aim 2, we will test the hyoothesis that the C/EBPalpha:p21 growth inhibitory pathway is deranged in old rats. Based on our preliminary results, we suggest that p21/SDI-1 level remains high in regenerating livers of old rats and this retention of p21/SDI-1 results, in the delay of DNA synthesis that has been described for old rats. Expression of p21/SDI-1 and other CDK inhibitors and proteins that regulate cell cycle division will be measured. In Specific Aim 3, we will study the molecular mechanisms of p21/SDI-1 regulation by C/EBPalpha. We have shown that in growth inhibited cells C/EBPalpha stabilized the p21/SDI-1 protein. Direct interaction of p21 and C/EBPalpha will be tested in vitro and in vivo using co-immunoprecipitation methods and the mammalian Matchmaker two-hybrid assay. If C/EBPalpha does not interact with p21/SDI-1 directly, we will test the possibility that stabilization occurs through regulation of genes whose products effect p21/SDI-1 stability. For this goal we will clone the genes that are responsible for C/EBPalpha-mediated growth arrest.